In a healthy mammal, the immune system protects the body from damage from foreign substances and pathogens. In some instances though, the immune system goes awry, producing traumatic insult and/or disease. For example, B-cells can produce antibodies that recognize self-proteins rather than foreign proteins, leading to the production of the autoantibodies characteristic of autoimmune diseases such as lupus erythematosus, rheumatoid arthritis, and the like. In other instances, the typically beneficial effect of the immune system in combating foreign materials is counterproductive, such as following organ transplantation. The power of the mammalian immune system, and in particular the human immune system, has been recognized and efforts have been made to control the system to avoid or ameliorate the deleterious consequences to health that result either from normal functioning of the immune system in an abnormal environment (e.g., organ transplantation) or from abnormal functioning of the immune system in an otherwise apparently normal environment (e.g., autoimmune disease progression). Additionally, efforts have been made to exploit the immune system to provide a number of target-specific diagnostic and therapeutic methodologies, relying on the capacity of antibodies to specifically recognize and bind antigenic targets with specificity.
One way in which the immune system protects the body is by production of specialized cells called B lymphocytes or B-cells. B-cells produce antibodies that bind to, and in some cases mediate destruction of, a foreign substance or pathogen. In some instances though, the human immune system, and specifically the B lymphocytes of the human immune system, go awry and disease results. There are numerous cancers that involve uncontrolled proliferation of B-cells. There are also numerous autoimmune diseases that involve B-cell production of antibodies that, instead of binding to foreign substances and pathogens, bind to parts of the body. In addition, there are numerous autoimmune and inflammatory diseases that involve B-cells in their pathology, for example, through inappropriate B-cell antigen presentation to T-cells or through other pathways involving B-cells. For example, autoimmune-prone mice deficient in B-cells do not develop autoimmune kidney disease, vasculitis or autoantibodies. (Shlomchik et al., J Exp. Med. 1994, 180:1295-306). Interestingly, these same autoimmune-prone mice which possess B-cells but are deficient in immunoglobulin production, do develop autoimmune diseases when induced experimentally (Chan et al., J Exp. Med. 1999, 189:1639-48), indicating that B-cells play an integral role in development of autoimmune disease.
B-cells can be identified by molecules on their cell surface. CD20 was the first human B-cell lineage-specific surface molecule identified by a monoclonal antibody. It is a non-glycosylated, hydrophobic 35 kDa B-cell transmembrane phosphoprotein that has both its amino and carboxy ends situated inside the cell. Einfeld et al., EMBO J. 1988, 7:711-17. CD20 is expressed by all normal mature B-cells, but is not expressed by precursor B-cells or plasma cells. Natural ligands for CD20 have not been identified, and the function of CD20 in B-cell biology is still incompletely understood.
Another B-cell lineage-specific cell surface molecule is CD37. CD37 is a heavily glycosylated 40-52 kDa protein that belongs to the tetraspanin transmembrane family of cell surface antigens. It traverses the cell membrane four times forming two extracellular loops and exposing its amino and carboxy ends to the cytoplasm. CD37 is highly expressed on normal antibody-producing (sIg+)B-cells, but is not expressed on pre-B-cells or plasma cells. The expression of CD37 on resting and activated T cells, monocytes and granulocytes is low and there is no detectable CD37 expression on NK cells, platelets or erythrocytes. See, Belov et al., Cancer Res., 61(11):4483-4489 (2001); Schwartz-Albiez et al., J. Immunol., 140(3): 905-914 (1988); and Link et al., J. Immunol., 137(9): 3013-3018 (1988). Besides normal B-cells, almost all malignancies of B-cell origin are positive for CD37 expression, including CLL, NHL, and hairy cell leukemia (Moore, et al. 1987; Merson and Brochier 1988; Faure, et al. 1990). CD37 participates in regulation of B-cell function, since mice lacking CD37 were found to have low levels of serum IgG1 and to be impaired in their humoral response to viral antigens and model antigens. It appears to act as a nonclassical costimulatory molecule or by directly influencing antigen presentation via complex formation with MHC class II molecules. See Knobeloch et al., Mol. Cell. Biol., 20(15):5363-5369 (2000).
Research and drug development has occurred based on the concept that B-cell lineage-specific cell surface molecules such as CD37 and CD20 can themselves be targets for antibodies that would bind to, and mediate destruction of, cancerous and autoimmune disease-causing B-cells that have CD37 and CD20 on their surfaces. Termed “immunotherapy,” antibodies made (or based on antibodies made) in a non-human animal that bind to CD37 or CD20 were given to a patient to deplete cancerous or autoimmune disease-causing B-cells.
Monoclonal antibody technology and genetic engineering methods have facilitated development of immunoglobulin molecules for diagnosis and treatment of human diseases. The domain structure of immunoglobulins is amenable to engineering, in that the antigen binding domains and the domains conferring effector functions may be exchanged between immunoglobulin classes and subclasses. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (1988). An extensive introduction as well as detailed information about all aspects of recombinant antibody technology can be found in the textbook “Recombinant Antibodies” (John Wiley & Sons, NY, 1999). A comprehensive collection of detailed antibody engineering lab Protocols can be found in R. Kontermann and S. Dübel (eds.), “The Antibody Engineering Lab Manual” (Springer Verlag, Heidelberg/New York, 2000).
An immunoglobulin molecule (abbreviated Ig), is a multimeric protein, typically composed of two identical light chain polypeptides and two identical heavy chain polypeptides (H2L2) that are joined into a macromolecular complex by interchain disulfide bonds, i.e., covalent bonds between the sulfhydryl groups of neighboring cysteine residues. Five human immunoglobulin classes are defined on the basis of their heavy chain composition, and are named IgG, IgM, IgA, IgE, and IgD. The IgG-class and IgA-class antibodies are further divided into subclasses, namely, IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively. Intrachain disulfide bonds join different areas of the same polypeptide chain, which results in the formation of loops that, along with adjacent amino acids, constitute the immunoglobulin domains. At the amino-terminal portion, each light chain and each heavy chain has a single variable region that shows considerable variation in amino acid composition from one antibody to another. The light chain variable region, VL, has a single antigen-binding domain and associates with the variable region of a heavy chain, VH (also containing a single antigen-binding domain), to form the antigen binding site of the immunoglobulin, the Fv.
In addition to variable regions, each of the full-length antibody chains has a constant region containing one or more domains. Light chains have a constant region containing a single domain. Thus, light chains have one variable domain and one constant domain. Heavy chains have a constant region containing several domains. The heavy chains in IgG, IgA, and IgD antibodies have three domains, which are designated CH1, CH2, and CH3; the heavy chains in IgM and IgE antibodies have four domains, CH1, CH2, CH3 and CH4. Thus, heavy chains have one variable domain and three or four constant domains. Noteworthy is the invariant organization of these domains in all known species, with the constant regions, containing one or more domains, being located at or near the C-terminus of both the light and heavy chains of immunoglobulin molecules, with the variable domains located towards the N-termini of the light and heavy chains. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (1988).
The heavy chains of immunoglobulins can also be divided into three functional regions: the Fd region (a fragment comprising VH and CH1, i.e., the two N-terminal domains of the heavy chain), the hinge region, and the Fc region (the “fragment crystallizable” region). The Fc region contains the domains that interact with immunoglobulin receptors on cells and with the initial elements of the complement cascade. Thus, the Fc region or fragment is generally considered responsible for the effector functions of an immunoglobulin, such as ADCC (antibody-dependent cell-mediated cytotoxicity), CDC (complement-dependent cytotoxicity) and complement fixation, binding to Fc receptors, greater half-life in vivo relative to a polypeptide lacking an FC region, protein A binding, and perhaps even placental transfer. Capon et al., Nature, 337: 525-531, (1989). Further, a polypeptide containing an Fc region allows for dimerization/multimerization of the polypeptide. These terms are also used for analogous regions of the other immunoglobulins.
Although all of the human immunoglobulin isotypes contain a recognizable structure in common, each isotype exhibits a distinct pattern of effector function. IgG, by way of nonexhaustive example, neutralizes toxins and viruses, opsonizes, fixes complement (CDC) and participates in ADCC. IgM, in contrast, neutralizes blood-borne pathogens and participates in opsonization. IgA, when associated with its secretory piece, is secreted and provides a primary defense to microbial infection via the mucosa; it also neutralizes toxins and supports opsonization. IgE mediates inflammatory responses, being centrally involved in the recruitment of other cells needed to mount a full response. IgD is known to provide an immunoregulatory function, controlling the activation of B cells. These characterizations of isotype effector functions provide a non-comprehensive illustration of the differences that can be found among human isotypes.
The hinge region, found in IgG, IgA, IgD, and IgE class antibodies, acts as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to the constant regions, the hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies among the IgG subclasses. The hinge region of IgG1 encompasses amino acids 216-231 and, because it is freely flexible, the Fab fragments can rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges. IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and four disulfide bridges. The hinge region of IgG2 lacks a glycine residue, is relatively short, and contains a rigid poly-proline double helix, stabilized by extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the IgG2 molecule. IgG3 differs from the other subclasses by its unique extended hinge region (about four times as long as the IgG1 hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix. In IgG3, the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility. The elongated hinge in IgG3 is also responsible for its higher molecular weight compared to the other subclasses. The hinge region of IgG4 is shorter than that of IgG1 and its flexibility is intermediate between that of IgG1 and IgG2. The flexibility of the hinge regions reportedly decreases in the order IgG3>IgG1>IgG4>IgG2. The four IgG subclasses also differ from each other with respect to their effector functions. This difference is related to differences in structure, including differences with respect to the interaction between the variable region, Fab fragments, and the constant Fc fragment.
According to crystallographic studies, the immunoglobulin hinge region can be further subdivided functionally into three regions: the upper hinge region, the core region, and the lower hinge region. Shin et al., 1992 Immunological Reviews 130:87. The upper hinge region includes amino acids from the carboxyl end of CH1 to the first residue in the hinge that restricts motion, generally the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the segmental flexibility of the antibody. The core hinge region contains the inter-heavy chain disulfide bridges, and the lower hinge region joins the amino terminal end of the CH2 domain and includes residues in CH2. Id. The core hinge region of human IgG1 contains the sequence Cys-Pro-Pro-Cys which, when dimerized by disulfide bond formation, results in a cyclic octapeptide believed to act as a pivot, thus conferring flexibility. The hinge region may also contain one or more glycosylation sites, which include a number of structurally distinct types of sites for carbohydrate attachment. For example, IgA1 contains five glycosylation sites within a 17-amino-acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, considered an advantageous property for a secretory immunoglobulin.
Conformational changes permitted by the structure and flexibility of the immunoglobulin hinge region polypeptide sequence may also affect the effector functions of the Fc portion of the antibody. Three general categories of effector functions associated with the Fc region include (1) activation of the classical complement cascade, (2) interaction with effector cells, and (3) compartmentalization of immunoglobulins. The different human IgG subclasses vary in the relative efficacies with which they fix complement, or activate and amplify the steps of the complement cascade. See, e.g., Kirschfink, 2001 Immunol. Rev. 180:177; Chakraborti et al., 2000 Cell Signal 12:607; Kohl et al., 1999 Mol. Immunol. 36:893; Marsh et al., 1999 Curr. Opin. Nephrol. Hypertens. 8:557; Speth et al., 1999 Wien Klin. Wochenschr. 111:378.
Exceptions to the H2L2 structure of conventional antibodies occur in some isotypes of the immunoglobulins found in camelids (camels, dromedaries and llamas; Hamers-Casterman et al., 1993 Nature 363:446; Nguyen et al., 1998 J. Mol. Biol. 275:413), nurse sharks (Roux et al., 1998 Proc. Nat. Acad. Sci. USA 95:11804), and in the spotted ratfish (Nguyen, et al., 2002 Immunogenetics 54(1):39-47). These antibodies can apparently form antigen-binding regions using only heavy chain variable region, i.e., these functional antibodies are homodimers of heavy chains only (referred to as “heavy-chain antibodies” or “HCAbs”). Despite the advantages of antibody technology in disease diagnosis and treatment, there are some disadvantageous aspects of developing whole-antibody technologies as diagnostic and/or therapeutic reagents. Whole antibodies are large protein structures exemplified by the heterotetrameric structure of the IgG isotype, containing two light and two heavy chains. Such large molecules are sterically hindered in certain applications. For example, in treatments of solid tumors, whole antibodies do not readily penetrate the interior of the tumor. Moreover, the relatively large size of whole antibodies presents a challenge to ensure that the in vivo administration of such molecules does not induce an immune response. Further, generation of active antibody molecules typically involves the culturing of recombinant eukaryotic cells capable of providing appropriate post-translational processing of the nascent antibody molecules, and such cells can be difficult to culture and difficult to induce in a manner that provides commercially useful yields of active antibody.
Recently, smaller immunoglobulin molecules have been constructed to overcome problems associated with whole immunoglobulin methodologies. A single-chain variable antibody fragment (scFv) comprises an antibody heavy chain variable domain joined via a short peptide to an antibody light chain variable domain (Huston et al., Proc. Natl. Acad. Sci. USA, 1988, 85: 5879-83). Because of the small size of scFv molecules, they exhibit more effective penetration into tissues than whole immunoglobulin. An anti-tumor scFv showed more rapid tumor penetration and more even distribution through the tumor mass than the corresponding chimeric antibody (Yokota et al., Cancer Res. 1992, 52:3402-08).
Despite the advantages that scFv molecules bring to serotherapy, several drawbacks to this therapeutic approach exist. An scFv is rapidly cleared from the circulation, which may reduce toxic effects in normal cells, but such rapid clearance impedes delivery of a minimum effective dose to the target tissue. Manufacturing adequate amounts of scFv for administration to patients has been challenging due to difficulties in expression and isolation of scFv that adversely affect the yield. During expression, scFv molecules lack stability and often aggregate due to pairing of variable regions from different molecules. Furthermore, production levels of scFv molecules in mammalian expression systems are low, limiting the potential for efficient manufacturing of scFv molecules for therapy (Davis et al, J Biol. Chem. 1990, 265:10410-18); Traunecker et al., EMBO J 1991, 10: 3655-59). Strategies for improving production have been explored, including addition of glycosylation sites to the variable regions (Jost, C. R. U.S. Pat. No. 5,888,773, Jost et al, J. Biol. Chem. 1994, 69: 26267-73).
Another disadvantage to using scFv for therapy is the lack of effector function. An scFv without a cytolytic function, such as the antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent-cytotoxicity (CDC) associated with the constant region of an immunoglobulin, may be ineffective for treating disease. Even though development of scFv technology began over 12 years ago, currently no scFv products are approved for therapy.
Alternatively, it has been proposed that fusion of an scFv to another molecule, such as a toxin, could take advantage of the specific antigen-binding activity and the small size of an scFv to deliver the toxin to a target tissue. Chaudary et al., Nature 1989, 339:394; Batra et al., Mol. Cell. Biol. 1991, 11:2200. Conjugation or fusion of toxins to scFvs has thus been offered as an alternative strategy to provide potent, antigen-specific molecules, but dosing with such conjugates or chimeras can be limited by excessive and/or non-specific toxicity due to the toxin moiety of such preparations. Toxic effects may include supraphysiological elevation of liver enzymes and vascular leak syndrome, and other undesired effects. In addition, immunotoxins are themselves highly immunogenic upon administration to a host, and host antibodies generated against the immunotoxin limit potential usefulness for repeated therapeutic treatments of an individual.
Nonsurgical cancer therapy, such as external irradiation and chemotherapy, can suffer from limited efficacy because of toxic effects on normal tissues and cells, due to the lack of specificity these treatments exhibit towards cancer cells. To overcome this limitation, targeted treatment methodologies have been developed to increase the specificity of the treatment for the cells and tissues in need thereof. An example of such a targeted methodology for in vivo use is the administration of antibody conjugates, with the antibody designed to specifically recognize a marker associated with a cell or tissue in need of treatment, and the antibody being conjugated to a therapeutic agent, such as a toxin in the case of cancer treatment. Antibodies, as systemic agents, circulate to sensitive and undesirable body compartments, such as the bone marrow. In acute radiation injury, destruction of lymphoid and hematopoietic compartments is a major factor in the development of septicemia and subsequent death. Moreover, antibodies are large, globular proteins that can exhibit poor penetration of tissues in need of treatment.
Human patients and non-human subjects suffering from a variety of end-stage disease processes frequently require organ transplantation. Organ transplantation, however, must contend with the untoward immune response of the recipient and guard against immunological rejection of the transplanted organ by depressing the recipient's cellular immune response to the foreign organ with cytotoxic agents which affect the lymphoid and other parts of the hematopoietic system. Graft acceptance is limited by the tolerance of the recipient to these cytotoxic chemicals, many of which are similar to the anticancer (antiproliferative) agents. Likewise, when using cytotoxic antimicrobial agents, particularly antiviral drugs, or when using cytotoxic drugs for autoimmune disease therapy, e.g., in treatment of systemic lupus erythematosis, a serious limitation is the toxic effects of the therapeutic agents on the bone marrow and the hematopoietic cells of the body.
Use of targeted therapies, such as targeted antibody conjugate therapy, is designed to localize a maximum quantity of the therapeutic agent at the site of desired action as possible, and the success of such therapies is revealed by the relatively high signal-to-background ratio of therapeutic agent. Examples of targeted antibodies include diagnostic or therapeutic agent conjugates of antibody or antibody fragments, cell- or tissue-specific peptides, and hormones and other receptor-binding molecules. For example, antibodies against different determinants associated with pathological and normal cells, as well as associated with pathogenic microorganisms, have been used for the detection and treatment of a wide variety of pathological conditions or lesions. In these methods, the targeting antibody is directly conjugated to an appropriate detecting or therapeutic agent as described, for example, in Hansen et al., U.S. Pat. No. 3,927,193 and Goldenberg, U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,460,459, 4,460,561, 4,624,846 and 4,818,709.
One problem encountered in direct targeting methods, i.e., in methods wherein the diagnostic or therapeutic agent (the “active agent”) is conjugated directly to the targeting moiety, is that a relatively small fraction of the conjugate actually binds to the target site, while the majority of conjugate remains in circulation and compromises in one way or another the function of the targeted conjugate. To ensure maximal localization of the active agent, an excess of the targeted conjugate is typically administered, ensuring that some conjugate will remain unbound and contribute to background levels of the active agent. A diagnostic conjugate, e.g., a radioimmunoscintigraphic or magnetic resonance imaging conjugate that does not bind its target can remain in circulation, thereby increasing background and decreasing resolution of the diagnostic technique. In the case of a therapeutic conjugate having a toxin as an active agent (e.g., a radioisotope, drug or toxic compound) attached to a long-circulating targeting moiety such as an antibody, circulating conjugate can result in unacceptable toxicity to the host, such as marrow toxicity or systemic side effects.
U.S. Pat. No. 4,782,840 discloses a method for reducing the effect of elevated background radiation levels during surgery. The method involves injection of a patient with antibodies specific for neoplastic tissue, with the antibodies labeled with radioisotopes having a suitably long half-life, such as Iodine-125. After injection of the radiolabeled antibody, the surgery is delayed at least 7-10 days, preferably 14-21 days, to allow any unbound radiolabeled antibody to be cleared to a low background level.
U.S. Pat. No. 4,932,412 discloses methods for reducing or correcting for non-specific background radiation during intraoperative detection. The methods include the administration to a patient who has received a radiolabeled primary antibody, of a contrast agent, subtraction agent or second antibody which binds the primary antibody.
Apart from producing the antibodies described above, the immune system includes a variety of cell types that have powerful biological effects. During hematopoiesis, bone marrow-derived stem cells differentiate into either mature cells of the immune system (“B” cells) or into precursors of cells that migrate out of the bone marrow to mature in the thymus (“T” cells).
B cells are central to the humoral component of an immune response. B cells are activated by an appropriate presentation of an antigen to become antibody-secreting plasma cells; antigen presentation also results in clonal expansion of the activated B cell. B cells are primarily responsible for the humoral component of an immune response. A plasma cell typically exhibits about 105 antibody molecules (IgD and IgM) on its surface.
T lymphocytes can be divided into two categories. The cytotoxic T cells, Tc lymphocytes or CTLs (CD8+ T cells), kill cells bearing foreign surface antigen in association with Class I MHC and can kill cells that are harboring intracellular parasites (either bacteria or viruses) as long as the infected cell is displaying a microbial antigen on its surface. Tc cells kill tumor cells and account for the rejection of transplanted cells. Tc cells recognize antigen-Class I MHC complexes on target cells, contact them, and release the contents of granules directly into the target cell membrane, which lyses the cell.
A second category of T cells is the helper T cell or Th lymphocyte (CD4+ T cells), which produces lymphokines that are “helper” factors in the maturation of B cells into antibody-secreting plasma cells. Th cells also produce certain lymphokines that stimulate the differentiation of effector T lymphocytes and the activity of macrophages. Th1 cells recognize antigen on macrophages in association with Class II MHC and become activated (by IL-1) to produce lymphokines, including the IFN-γ that activates macrophages and NK cells. These cells mediate various aspects of the cell-mediated immunity response including delayed-type hypersensitivity reactions. Th2 cells recognize antigen in association with Class II MHC on an antigen presenting cell or APC (e.g., migratory macrophages and dendritic cells) and then produce interleukins and other substances that stimulate specific B-cell and T-cell proliferation and activity.
Beyond serving as APCs that initiate T cell interactions, development, and proliferation, macrophages are involved in expression of cell-mediated immunity because they become activated by IFN-γ produced in a cell-mediated immune response. Activated macrophages have increased phagocytic potential and release soluble substances that cause inflammation and destroy many bacteria and other cells. Natural Killer cells are cytotoxic cells that lyse cells bearing new antigen, regardless of their MHC type, and even lyse some cells that bear no MHC proteins. Natural Killer T cells, or NK cells, are defined by their ability to kill cells displaying a foreign antigen (e.g., tumor cells), regardless of MHC type, and regardless of previous sensitization (exposure) to the antigen. NK cells can be activated by IL-2 and IFN-γ, and lyse cells in the same manner as cytotoxic T lymphocytes. Some NK cells have receptors for the Fc domain of the IgG antibody (e.g, CD16 or FCγRIII) and are thus able to bind to the Fc portion of IgG on the surface of a target cell and release cytolytic components that kill the target cell via antibody-dependent cell-mediated cytotoxicity.
Another group of cells is the granulocytes or polymorphonuclear leukocytes (PMNs). Neutrophils, one type of PMN, kill bacterial invaders and phagocytose the remains. Eosinophils are another type of PMN and contain granules that prove cytotoxic when released upon another cell, such as a foreign cell. Basophils, a third type of PMN, are significant mediators of powerful physiological responses (e.g., inflammation) that exert their effects by releasing a variety of biologically active compounds, such as histamine, serotonin, prostaglandins, and leukotrienes. Common to all of these cell types is the capacity to exert a physiological effect within an organism, frequently by killing, and optionally scavenging, deleterious compositions such as foreign cells.
Although a variety of mammalian cells, including cells of the immune system, are capable of directly exerting a physiological effect (e.g., cell killing, typified by Tc, NK, some PMN, macrophage, and the like), other cells indirectly contribute to a physiological effect. For example, initial presentation of an antigen to a naïve T cell of the immune system requires MHC presentation that mandates cell-cell contact. Further, there often needs to be contact between an activated T cell and an antigen-specific B cell to obtain a particular immunogenic response. A third form of cell-cell contact often seen in immune responses is the contact between an activated B cell and follicular dendritic cells. Each of these cell-cell contact requirements complicates the targeting of a biologically active agent to a given target.
Complement-dependent cytotoxicity (CDC) is believed to be a significant mechanism for clearance of specific target cells such as tumor cells. CDC is a series of events that consists of a collection of enzymes that become activated by each other in a cascade fashion. Complement has an important role in clearing antigen, accomplished by its four major functions: (1) local vasodilation; (2) attraction of immune cells, especially phagocytes (chemotaxis); (3) tagging of foreign organisms for phagocytosis (opsonization); and (4) destruction of invading organisms by the membrane attack complex (MAC attack). The central molecule is the C3 protein. It is an enzyme that is split into two fragments by components of either the classical pathway or the alternative pathway. The classical pathway is induced by antibodies, especially IgG and IgM, while the alternative pathway is nonspecifically stimulated by bacterial products like lipopolysaccharide (LPS). Briefly, the products of the C3 split include a small peptide C3a which is chemotactic for phagocytic immune cells and results in local vasodilation by causing the release of C5a fragment from C5. The other part of C3, C3b, coats antigens on the surface of foreign organisms and acts to opsonize the organism for destruction. C3b also reacts with other components of the complement system to form an MAC consisting of C5b, C6, C7, C8 and C9.
There are problems associated with the use of antibodies in human therapy because the response of the immune system to any antigen, even the simplest, is “polyclonal,” i.e., the system manufactures antibodies of a great range of structures both in their binding regions as well as in their effector regions.
Two approaches have been used in an attempt to reduce the problem of immunogenic antibodies. The first is the production of chimeric antibodies in which the antigen-binding part (variable regions) of a mouse monoclonal antibody is fused to the effector part (constant region) of a human antibody. In a second approach, antibodies have been altered through a technique known as complementarity determining region (CDR) grafting or “humanization.” This process has been further improved to include changes referred to as “reshaping” (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann, et al., 1988 Nature 332:323-337; Tempest, et al., Bio/Technol 1991 9:266-271), “hyperchimerization” (Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol 148:1149-1154), and “veneering” (Mark, et al., In: Metcalf B W, Dalton B J, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994:291-312).
An average of less than one therapeutic antibody per year has been introduced to the market beginning in 1986, eleven years after the publication of monoclonal antibodies. Five murine monoclonal antibodies were introduced into human medicine over a ten year period from 1986-1995, including “muromonab-CD3” (OrthoClone OKT3®) for acute rejection of organ transplants; “edrecolomab” (Panorex®) for colorectal cancer; “odulimomab” (Antilfa®) for transplant rejection; and, “ibritumomab” (Zevalin® yiuxetan) for non-Hodgkin's lymphoma. Additionally, a monoclonal Fab, “abciximab” (ReoPro®) has been marketed for preventing coronary artery reocclusion. Three chimeric monoclonal antibodies were also launched: “rituximab” (Rituxan®) for treating B cell lymphomas; “basiliximab” (Simulect®) for transplant rejection; and “infliximab” (Remicade®) for treatment of rheumatoid arthritis and Crohn's disease. Additionally, “abciximab” (ReoPro®), a 47.6 kD Fab fragment of a chimeric human-murine monoclonal antibody is marketed as an adjunct to percutaneous coronary intervention for the prevention of cardiac ischemic complications in patients undergoing percutaneous coronary intervention. Finally, seven “humanized” monoclonal antibodies have been launched. “Daclizumab” (Zenapax®) is used to prevent acute rejection of transplanted kidneys; “palivizumab” (Synagis®) for RSV; “trastuzumab” (Herceptin®) binds HER-2, a growth factor receptor found on breast cancers cells; “gemtuzumab” (Mylotarg®) for acute myelogenous leukemia (AML); and “alemtuzumab” (MabCampath®) for chronic lymphocytic leukemia; “adalimumab” (Humira® (D2E7)) for the treatment of rheumatoid arthritis; and, “omalizumab” (Xolair®), for the treatment of persistent asthma.
Thus, a variety of antibody technologies have received attention in the effort to develop and market more effective therapeutics and palliatives. Unfortunately, problems continue to compromise the promise of each of these therapies. For example, the majority of cancer patients treated with rituximab relapse, generally within about 6-12 months, and fatal infusion reactions within 24 hours of rituximab infusion have been reported. Acute renal failure requiring dialysis with instances of fatal outcome has also been reported in treatments with rituximab, as have severe, occasionally fatal, mucocutaneous reactions. Additionally, high doses of rituximab are required for intravenous injection because the molecule is large, approximately 150 kDa, and diffusion into the lymphoid tissues, where many tumor cells may reside is limited.
Trastuzumab administration can result in the development of ventricular dysfunction, congestive heart failure, and severe hypersensitivity reactions (including anaphylaxis), infusion reactions, and pulmonary events. Daclizumab immunosuppressive therapy poses an increased risk for developing lymphoproliferative disorders and opportunistic infections. Death from liver failure, arising from severe hepatotoxicity, and from veno-occlusive disease (VOD), has been reported in patients who received gemtuzumab.
Hepatotoxicity was also reported in patients receiving alemtuzumab. Serious and, in some rare instances fatal, pancytopenia/marrow hypoplasia, autoimmune idiopathic thrombocytopenia, and autoimmune hemolytic anemia have occurred in patients receiving alemtuzumab therapy. Alemtuzumab can also result in serious infusion reactions as well as opportunistic infections. In patients treated with adalimumab, serious infections and sepsis, including fatalities, have been reported, as has the exacerbation of clinical symptoms and/or radiographic evidence of demyelinating disease, and patients treated with adalimumab in clinical trials had a higher incidence of lymphoma than the expected rate in the general population. Omalizumab reportedly induces malignancies and anaphylaxis.
Cancer includes a broad range of diseases, affecting approximately one in four individuals worldwide. Rapid and unregulated proliferation of malignant cells is a hallmark of many types of cancer, including hematological malignancies. Although patients with a hematologic malignant condition have benefited from advances in cancer therapy in the past two decades, Multani et al., 1998 J. Clin. Oncology 16:3691-3710, and remission times have increased, most patients still relapse and succumb to their disease. Barriers to cure with cytotoxic drugs include, for example, tumor cell resistance and the high toxicity of chemotherapy, which prevents optimal dosing in many patients.
Treatment of patients with low grade or follicular B cell lymphoma using a chimeric CD20 monoclonal antibody has been reported to induce partial or complete responses in patients. McLaughlin et al., 1996 Blood 88:90a (abstract, suppl. 1); Maloney et al., 1997 Blood 90:2188-95. However, as noted above, tumor relapse commonly occurs within six months to one year. Further improvements in serotherapy are needed to induce more durable responses, for example, in low grade B cell lymphoma, and to allow effective treatment of high grade lymphoma and other B cell diseases.
Another approach has been to target radioisotopes to B cell lymphomas using monoclonal antibodies specific for CD20. While the effectiveness of therapy is reportedly increased, associated toxicity from the long in vivo half-life of the radioactive antibody increases, sometimes requiring that the patient undergo stem cell rescue. Press et al., 1993 N. Eng. J. Med. 329:1219-1224; Kaminski et al., 1993 N. Eng. J. Med. 329:459-65. Monoclonal antibodies to CD20 have also been cleaved with proteases to yield F(ab′)2 or Fab fragments prior to attachment of radioisotope. This has been reported to improve penetration of the radioisotope conjugate into the tumor and to shorten the in vivo half-life, thus reducing the toxicity to normal tissues. However, these molecules lack effector functions, including complement fixation and/or ADCC.
Autoimmune diseases include autoimmune thyroid diseases, which include Graves' disease and Hashimoto's thyroiditis. In the United States alone, there are about 20 million people who have some form of autoimmune thyroid disease. Autoimmune thyroid disease results from the production of autoantibodies that either stimulate the thyroid to cause hyperthyroidism (Graves' disease) or destroy the thyroid to cause hypothyroidism (Hashimoto's thyroiditis). Stimulation of the thyroid is caused by autoantibodies that bind and activate the thyroid stimulating hormone (TSH) receptor. Destruction of the thyroid is caused by autoantibodies that react with other thyroid antigens. Current therapy for Graves' disease includes surgery, radioactive iodine, or antithyroid drug therapy. Radioactive iodine is widely used, since antithyroid medications have significant side effects and disease recurrence is high. Surgery is reserved for patients with large goiters or where there is a need for very rapid normalization of thyroid function. There are no therapies that target the production of autoantibodies responsible for stimulating the TSH receptor. Current therapy for Hashimoto's thyroiditis is levothyroxine sodium, and lifetime therapy is expected because of the low likelihood of remission. Suppressive therapy has been shown to shrink goiters in Hashimoto's thyroiditis, but no therapies that reduce autoantibody production to target the disease mechanism are known.
Rheumatoid arthritis (RA) is a chronic disease characterized by inflammation of the joints, leading to swelling, pain, and loss of function. RA affects an estimated 2.5 million people in the United States. RA is caused by a combination of events including an initial infection or injury, an abnormal immune response, and genetic factors. While autoreactive T cells and B cells are present in RA, the detection of high levels of antibodies that collect in the joints, called rheumatoid factor, is used in the diagnosis of RA. Current therapy for RA includes many medications for managing pain and slowing the progression of the disease. No therapy has been found that can cure the disease. Medications include nonsteroidal anti-inflammatory drugs (NSAIDS), and disease modifying anti-rheumatic drugs (DMARDS). NSAIDS are useful in benign disease, but fail to prevent the progression to joint destruction and debility in severe RA. Both NSAIDS and DMARDS are associated with significant side effects. Only one new DMARD, Leflunomide, has been approved in over 10 years. Leflunomide blocks production of autoantibodies, reduces inflammation, and slows progression of RA. However, this drug also causes severe side effects including nausea, diarrhea, hair loss, rash, and liver injury.
Systemic Lupus Erythematosus (SLE) is an autoimmune disease caused by recurrent injuries to blood vessels in multiple organs, including the kidney, skin, and joints. SLE is estimated to affect over 500,000 people in the United States. In patients with SLE, a faulty interaction between T cells and B cells results in the production of autoantibodies that attack the cell nucleus. These include anti-double stranded DNA and anti-Sm antibodies. Autoantibodies that bind phospholipids are also found in about half of SLE patients, and are responsible for blood vessel damage and low blood counts. Immune complexes accumulate in the kidneys, blood vessels, and joints of SLE patients, where they cause inflammation and tissue damage. No treatment for SLE has been found to cure the disease. NSAIDS and DMARDS are used for therapy depending upon the severity of the disease. Plasmapheresis with plasma exchange to remove autoantibodies can cause temporary improvement in SLE patients. There is general agreement that autoantibodies are responsible for SLE, so new therapies that deplete the B cell lineage, allowing the immune system to reset as new B cells are generated from precursors, would offer hope for long lasting benefit in SLE patients.
Sjogren's syndrome is an autoimmune disease characterized by destruction of the body's moisture-producing glands. Sjogren's syndrome is one of the most prevalent autoimmune disorders, striking up to an estimated 4 million people in the United States. About half of the people stricken with Sjogren's syndrome also have a connective tissue disease, such as RA, while the other half have primary Sjogren's syndrome with no other concurrent autoimmune disease. Autoantibodies, including anti-nuclear antibodies, rheumatoid factor, anti-fodrin, and anti-muscarinic receptor are often present in patients with Sjogren's syndrome. Conventional therapy includes corticosteroids, and additional more effective therapies would be of benefit.
Immune thrombocytopenic purpura (ITP) is caused by autoantibodies that bind to blood platelets and cause their destruction. Some cases of ITP are caused by drugs, and others are associated with infection, pregnancy, or autoimmune disease such as SLE. About half of all cases are classified as being of idiopathic origin. The treatment of ITP is determined by the severity of the symptoms. In some cases, no therapy is needed although in most cases immunosuppressive drugs, including corticosteroids or intravenous infusions of immune globulin to deplete T cells, are provided. Another treatment that usually results in an increased number of platelets is removal of the spleen, the organ that destroys antibody-coated platelets. More potent immunosuppressive drugs, including cyclosporine, cyclophosphamide, or azathioprine are used for patients with severe cases. Removal of autoantibodies by passage of patients' plasma over a Protein A column is used as a second line treatment in patients with severe disease. Additional more effective therapies are needed.
Multiple sclerosis (MS) is also an autoimmune disease. It is characterized by inflammation of the central nervous system and destruction of myelin, which insulates nerve cell fibers in the brain, spinal cord, and body. Although the cause of MS is unknown, it is widely believed that autoimmune T cells are primary contributors to the pathogenesis of the disease. However, high levels of antibodies are present in the cerebrospinal fluid of patients with MS, and some predict that the B cell response leading to antibody production is important for mediating the disease. No B cell depletion therapies have been studied in patients with MS, and there is no cure for MS. Current therapy is corticosteroids, which can reduce the duration and severity of attacks, but do not affect the course of MS over time. New biotechnology interferon (IFN) therapies for MS have recently been approved but additional more effective therapies are required.
Myasthenia Gravis (MG) is a chronic autoimmune neuromuscular disorder that is characterized by weakness of the voluntary muscle groups. MG affects about 40,000 people in the United States. MG is caused by autoantibodies that bind to acetylcholine receptors expressed at neuromuscular junctions. The autoantibodies reduce or block acetylcholine receptors, preventing the transmission of signals from nerves to muscles. There is no known cure for mg. Common treatments include immunosuppression with corticosteroids, cyclosporine, cyclophosphamide, or azathioprine. Surgical removal of the thymus is often used to blunt the autoimmune response. Plasmapheresis, used to reduce autoantibody levels in the blood, is effective in mg, but is short-lived because the production of autoantibodies continues. Plasmapheresis is usually reserved for severe muscle weakness prior to surgery. New and effective therapies would be of benefit.
Psoriasis affects approximately five million people, and is characterized by autoimmune inflammation in the skin. Psoriasis is also associated with arthritis in 30% (psoriatic arthritis). Many treatments, including steroids, uv light retinoids, vitamin D derivatives, cyclosporine, and methotrexate have been used but it is also clear that psoriasis would benefit from new and effective therapies. Scleroderma is a chronic autoimmune disease of the connective tissue that is also known as systemic sclerosis. Scleroderma is characterized by an overproduction of collagen, resulting in a thickening of the skin, and approximately 300,000 people in the United States have scleroderma, which would also benefit from new and effective therapies.
Apparent from the foregoing discussion are needs for improved compositions and methods to treat, ameliorate or prevent a variety of diseases, disorders and conditions, including cancer and autoimmune diseases.